SUMMARY OF WORK Excitation-contraction coupling in the heart is a prototype for intracellular signaling by spatially and temporally localized calcium gradients. Contraction is triggered by calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, via calcium release channels commonly known as ryanodine receptors (RyR's), but the details of the process, and the way in which RyR's are coupled to the calcium microdomains produced by sarcolemmal channels are uncertain. We are developing a model system in which cardiac myocytes are differentiated in vitro from mouse embryonic stem cells. This will permit genetic manipulations of the channels and the signals which control their localization, using homologous recombination gene targeting methods. As a first step, we have begun to characterize the EC coupling process in these cardiac myocytes. It has generally been believed that excitation-contraction coupling in pre-natal and neonatal cardiac muscle occurs primarily by entry of calcium through sarcolemmal calcium channels, with SR calcium release making a minor contribution. Embryonic stem cells (R-1) and embryonic carcinoma cells (P-19) were cultured under conditions developed by Wobus et al which favor formation of embryoid bodies and differentiation of a cardiac lineage. Spontaneously contracting cells were excised from embryoid bodies, loaded with the fluorescent calcium probe fluo-3, and studied on the stage of a confocal microscope. We observed frequent, spontaneous calcium sparks and waves, which are the hallmark of CICR, in cells as early as the seventh developmental day. This indicates that these cells are a viable model in which to study the SR calcium release machinery.